8  Microglial subpopulations

Identification of Microglial Subpopulations

Published

August 9, 2025

8.1 UMAPs

8.1.1 All cell types

Comparison of UMAPs previously annotated using ScType and clustered with Seurat.

8.1.2 Only microglia

Ribosomal Content Varies Across Microglial Subclusters. However, when examining the top marker genes obtained from FindAllMarkers, there are no ribosomal genes among the most discriminative features (except in cluster 17). This suggests that while ribosomal content varies, it does not directly drive cluster identity at the transcriptional marker level.

8.1.3 Clusters according to treatments (Adu vs. IgG)

All those cluster appear in both treatment groups:

Number of cells per cluster
seurat_clusters Adu IgG
0 3546 2987
1 2568 2343
2 3103 1787
4 2159 1540
6 1732 1210
8 1335 968
10 1259 912
12 1125 585
14 611 364
15 510 390
17 345 336
22 253 123
23 132 163
27 93 51

8.2 Microglial subpopulations. Cluster-specific markers.

The FindMarkers function was applied to the complete dataset, analyzing all available cells without any subsetting. By not subsetting prior to marker detection, the analysis maximizes statistical power.

8.2.1 Outside the big blob

8.2.1.1 Cluster 22

Gene Broad Class Finer Subtype Notes
Mrc1 Microglia / Macrophage Perivascular macrophages CD206; enriched in border-associated macrophages (BAMs) and perivascular/meningeal macrophages.
F13a1 Microglia / Macrophage Perivascular macrophages Co-expressed with Mrc1; clotting factor XIII subunit A.
Ms4a6c Microglia Homeostatic microglia Member of MS4A family; enriched in microglia under steady-state conditions.
Pf4 Microglia / Macrophage Border-associated macrophages Also known as platelet factor 4; found in BAM-like cells.
Pid1 Microglia Activated microglia Linked to metabolic regulation and activation states.
Dab2 Microglia / Macrophage BAM-like macrophages Endocytic adaptor; enriched in perivascular/meningeal macrophages.
Ctsc Myeloid (Microglia / Macrophage) Activated microglia/macrophages Cathepsin C; lysosomal protease.
Stab1 Microglia / Macrophage Perivascular macrophages Scavenger receptor; strong BAM marker.
Ccl7 Microglia / Macrophage Activated/chemotactic state Chemokine induced by inflammation.
Pim1 Microglia Activated microglia Serine/threonine kinase; inflammatory regulation.
Rnf150 Microglia Homeostatic microglia E3 ubiquitin ligase; enriched in resting microglia.
Ifitm2 Myeloid / Endothelial Interferon-stimulated microglia Broad interferon response gene; can be endothelial too.
Iqgap1 Broad Ubiquitous but higher in activated microglia & astrocytes Scaffold protein; upregulated in injury.
Maf Microglia / Macrophage BAM-like macrophages Transcription factor important for BAM identity.
Ccl8 Microglia / Macrophage Activated state Chemokine; inflammatory signature.
Klf6 Microglia / Astrocyte Activated microglia/astrocytes Injury-induced transcription factor.
Cebpb Microglia / Astrocyte Inflammatory state Regulates reactive glia.
Man1a Broad Housekeeping Golgi alpha-mannosidase; not cell-type specific.
Cytip Microglia / Macrophage BAM-like macrophages Cytokine-induced protein in myeloid cells.
Srgn Microglia / Macrophage Activated myeloid cells Proteoglycan; high in inflammatory myeloid cells.

Almost all these genes map to myeloid lineage in the brain, especially border-associated macrophages (BAMs) — which include perivascular, meningeal, and choroid plexus macrophages — and to a lesser extent activated microglia. The signature is strongly enriched for perivascular macrophage markers (Mrc1, F13a1, Stab1, Pf4, Maf, Dab2) combined with inflammatory chemokines (Ccl7, Ccl8, Cebpb).

8.2.1.2 Cluster 23

Gene Broad Class Finer Subtype Notes
Ndufa4l2 Endothelial Capillary / hypoxia-responsive EC Mitochondrial complex I subunit; enriched in brain endothelial cells under low oxygen.
Egflam Endothelial / Pericyte Vascular leptomeningeal cells Extracellular matrix glycoprotein; strong in vascular interfaces.
Gucy1b1 Endothelial Arterial/venous EC Soluble guanylate cyclase β1 subunit.
Il34 Neuron (source) / Microglia (target receptor CSF1R) Secreted trophic factor Neurons produce IL-34 to support microglia survival; expression in cortical excitatory neurons.
Arhgap42 Endothelial / Smooth muscle Vascular SMC/pericytes Rho GTPase-activating protein.
Galnt17 Endothelial / Pericyte Vascular interface Glycosyltransferase; enriched in mural cells.
Lin7a Neuron Excitatory neuron (layer-specific) Synaptic scaffolding protein.
Ctdspl Neuron Broad neuronal expression Transcriptional repressor; high in excitatory neurons.
P2ry14 Endothelial / Pericyte Venous EC / pericytes UDP-glucose receptor; enriched in vascular cells.
Sntb1 Neuron / Glia Synaptic protein Found in neurons and astrocytic endfeet; part of dystrophin complex.
Art3 Endothelial / Pericyte Vascular smooth muscle-like ADP-ribosyltransferase; mural cell enrichment.
Airn Endothelial Capillary EC (non-coding RNA) Imprinted lncRNA; vascular expression.
Myl9 Smooth muscle / Pericyte Contractile pericytes Myosin light chain 9.
Ecm2 Endothelial / Pericyte Extracellular matrix of vasculature ECM component; vascular specificity.
Nbl1 Endothelial Vascular niche BMP antagonist; enriched in EC.
Septin11 Broad Cytoskeletal; ubiquitous Not cell-type specific; structural role.
Kcnj8 Pericyte / SMC Contractile mural cells Potassium inward rectifier channel Kir6.1.
Cspg4 Pericyte / OPC NG2+ cells Canonical marker of pericytes and oligodendrocyte precursor cells.
Ddit4l Endothelial / Astrocyte Hypoxia-stress responsive Regulates mTOR; induced in vascular and glial cells under stress.
Morrbid Myeloid (perivascular macrophages) Apoptosis regulator lncRNA in tissue-resident myeloid cells; in brain, enriched in BAMs.

This set is dominated by vascular-associated cells — mostly endothelial cells and mural cells (pericytes, vascular smooth muscle cells) — with a few neuronal genes (Il34, Lin7a, Ctdspl), one perivascular macrophage gene (Morrbid), and a dual-identity pericyte/OPC marker (Cspg4). The strong enrichment for Ndufa4l2, P2ry14, Kcnj8, Myl9 points to capillary/perivascular structures, often at the brain–blood barrier.

8.2.1.3 Cluster 15

Gene Broad Class Finer Subtype Notes
Itk Immune (T cell) T lymphocytes IL-2–inducible T-cell kinase; canonical T-cell signaling.
Cd3g Immune (T cell) T lymphocytes CD3 complex γ chain; TCR signaling.
Cd3e Immune (T cell) T lymphocytes CD3 complex ε chain; core T-cell receptor component.
Grap2 Immune (T cell/NK) T cells, NK cells Adaptor in TCR/NK signaling.
Cxcr6 Immune (T cell) Tissue-resident memory T cells Chemokine receptor guiding brain TRM infiltration.
Themis Immune (T cell) T lymphocytes Thymocyte development protein.
Gimap4 Immune (T cell) T lymphocytes Small GTPase enriched in T cells.
Lck Immune (T cell/NK) TCR/NK signaling Src-family kinase in TCR signaling.
Trac Immune (T cell) αβ T lymphocytes TCR α constant region.
Cd226 Immune (T cell/NK) Cytotoxic T cells, NK cells Co-stimulatory receptor.
Stat4 Immune (T cell/NK) Th1 CD4⁺ T cells, NK cells Transcription factor in IL-12 signaling.
Atp8b4 Microglia / Myeloid Homeostatic microglia Phospholipid transporter; strong microglial marker in brain.
Ikzf3 Immune (B cell/T cell) Memory B cells, T cells Aiolos transcription factor.
Ctsw Immune (T cell/NK) Cytotoxic lymphocytes Cathepsin W; granule protein in CD8⁺ T cells and NK cells.
Runx3 Immune (T cell) CD8⁺ cytotoxic T cells Lineage-defining transcription factor.
Adam19 Broad Various Metalloproteinase; expressed in multiple lineages, higher in vascular cells.
Pde7a Immune (T cell) T lymphocytes cAMP phosphodiesterase enriched in T cells.
Gramd3 Broad Limited immune association Poorly characterized; low-level in immune and vascular cells.
Bcl2 Broad Anti-apoptotic Expressed in many cell types; survival factor in lymphocytes.
Ptprc (CD45) Immune (pan-leukocyte) All immune cells Canonical pan-leukocyte marker.

This list is overwhelmingly T-cell–specific, with strong enrichment for CD3-complex genes (Cd3e, Cd3g, Trac), TCR signaling molecules (Itk, Lck, Themis, Grap2), and effector/memory T cell markers (Cxcr6, Runx3, Cd226, Stat4). These signatures correspond to brain-infiltrating T lymphocytes, especially CD8⁺ cytotoxic and tissue-resident memory (TRM) subsets, with a possible small contribution from NK cells.

One gene (Atp8b4) is clearly a microglia marker, suggesting either minor contamination or genuine presence of myeloid cells in this population. Ptprc confirms leukocyte origin.

8.2.1.4 Cluster 17

Gene Broad Class Finer Subtype Notes
Syngr1 Neuron Broad neuronal (synaptic vesicle protein) Pan-synaptic marker; in both excitatory and inhibitory neurons.
Tyrobp Microglia / Macrophage Activated microglia, DAM (disease-associated microglia) Adaptor protein in TREM2 signaling.
Fau Broad Housekeeping Ribosomal protein; not cell-type specific.
Rplp1 Broad Housekeeping Ribosomal large subunit protein; ubiquitous.
Tmsb4x Broad (with immune bias) Cytoskeleton/actin regulator Expressed in many cells; high in microglia and astrocytes.
Fcer1g Microglia / Macrophage DAM/activated microglia Immunoreceptor adaptor in TREM2–TYROBP axis.
Trem2 Microglia DAM signature Alzheimer’s and injury-associated microglia.
Cst3 Microglia / Astrocyte Broad glial Cystatin C; high in microglia, astrocytes, and choroid plexus epithelium.
Cd52 Immune (lymphoid/myeloid) Lymphocytes, some microglia GPI-anchored protein; leukocyte marker with low microglial expression.
C1qc Microglia Homeostatic microglia Complement subcomponent; part of C1q complex.
Ctsd Microglia / Broad glia Lysosomal protease High in microglia and astrocytes.
C1qa Microglia Homeostatic microglia Complement component; C1q complex.
Ctsz Microglia Activated microglia Lysosomal protease upregulated in activation.
C1qb Microglia Homeostatic microglia Complement C1q complex.
Ctsb Microglia / Broad glia Lysosomal protease Ubiquitous in glia; higher in microglia.
Hexb Microglia Canonical microglia marker Beta-hexosaminidase subunit; stable homeostatic marker.
Cox6a2 Neuron Oxidative phosphorylation, neuronal subtype Mitochondrial protein; often higher in fast-spiking neurons.
Grn Microglia / Astrocyte Activated microglia Progranulin; injury/inflammation marker.
Fxyd5 Broad (Glia bias) Astrocytes, microglia Cell adhesion regulator.
Mpeg1 Microglia / Macrophage BAMs and activated microglia Perforin-like protein; high in phagocytic myeloid cells.

This list is dominated by microglia, with a strong homeostatic core signature (C1qa, C1qb, C1qc, Hexb) combined with activation/DAM markers (Trem2, Tyrobp, Fcer1g, Grn, Mpeg1). This suggests you are seeing a mixed microglial population — mostly homeostatic, with a substantial activated/DAM subset.

A few genes are neuronal (Syngr1, Cox6a2) or ubiquitous ribosomal (Fau, Rplp1), which could represent low-level neuronal contamination or ambient RNA.

8.2.1.5 Cluster 14

Gene Broad Class Finer Subtype Notes
Ifi211 Myeloid (microglia/macrophage) Interferon-stimulated microglia Part of IFN-inducible family.
Ifit3 Myeloid (microglia/macrophage) Type I IFN response Canonical antiviral gene.
Mx1 Myeloid (microglia/macrophage) IFN-stimulated antiviral state Dynamin-like GTPase; viral defense.
Ifi209 Myeloid IFN-stimulated microglia AIM2-like receptor family.
Ifit2 Myeloid IFN-induced microglia Antiviral IFN response protein.
A330040F15Rik Myeloid IFN-response Predicted gene; co-expressed with ISGs.
Oasl2 Myeloid IFN-stimulated antiviral 2’-5’-oligoadenylate synthetase-like.
Rsad2 (Viperin) Myeloid Antiviral IFN response Strong ISG in microglia/macrophages.
Ifi204 Myeloid IFN/DNA-sensing microglia p204 protein, innate immunity.
Gm4951 Myeloid IFN-induced Predicted; co-expression with ISGs.
Ccl12 Myeloid Activated inflammatory macrophages Monocyte chemoattractant.
Parp14 Myeloid IFN-stimulated ADP-ribosyltransferase, antiviral role.
Herc6 Myeloid IFN-induced antiviral E3 ligase in ISGylation pathway.
Ifih1 Myeloid Viral RNA sensing microglia MDA5 helicase; innate immune sensor.
Bst2 Myeloid IFN-induced microglia/endothelial Tetherin; antiviral restriction factor.
Pik3ap1 Myeloid / B cell B cell receptor adaptor; some myeloid expression Possible infiltrating lymphoid origin.
Cxcl10 Myeloid IFN-induced chemokine T cell chemoattractant (IP-10).
Lyn Myeloid / B cell Src-family kinase in immune signaling Present in microglia and B cells.
Slc15a3 Myeloid IFN-induced microglia/macrophages Lysosomal peptide transporter; innate immunity.
Hpse Myeloid / Endothelial Activated immune cells Heparanase; ECM remodeling during inflammation.

This is a Type I interferon–driven microglial/macrophage activation signature with strong antiviral and inflammatory chemokine components. Key canonical ISGs (Ifit3, Mx1, Rsad2, Oasl2, Cxcl10) are all upregulated in microglia during viral infection models, neuroinflammation, or interferonopathies.

A few genes (Pik3ap1, Lyn) have higher expression in B cells, so there might be a minor infiltrating B-lymphocyte component, but the dominant source here is clearly microglia in an interferon-activated state.

8.2.2 Inside the big blob

8.2.2.1 Cluster 0

Gene Broad Class Finer Subtype Notes
Nfkbiz Myeloid (microglia/macrophage) NF-κB–responsive microglia Nuclear IκB family; inflammation-induced.
Bcl2a1b Myeloid Pro-survival inflammatory myeloid cells Anti-apoptotic gene; NF-κB target.
Tnf Myeloid Activated microglia/macrophages Key pro-inflammatory cytokine.
Ier3 Myeloid Stress-response microglia Immediate early gene; injury-induced.
Bcl2a1d Myeloid Pro-survival inflammatory myeloid cells Paralogue of Bcl2a1b.
Il1b Myeloid Activated inflammasome+ microglia Pro-inflammatory cytokine; NLRP3-dependent.
Mapkapk2 Myeloid Stress/inflammation response p38 MAPK-activated protein kinase.
Cd14 Myeloid Classical monocytes / activated microglia LPS co-receptor; innate immune sensing.
Mir155hg Myeloid Activated inflammatory microglia Host gene for miR-155; immune activation.
Nlrp3 Myeloid Inflammasome+ activated microglia Inflammasome sensor; IL-1β maturation.
Rel Myeloid NF-κB transcription factor Drives cytokine production.
Dennd4a Broad Vesicular trafficking Not cell-type specific; present in many cells.
Cd86 Myeloid Antigen-presenting activated microglia Co-stimulatory molecule for T cell activation.
Osm (Oncostatin M) Myeloid Activated myeloid cells Cytokine; can signal to astrocytes/endothelium.
Osbpl8 Broad Lipid transport Housekeeping; not specific.
Tnfsf9 (4-1BBL) Myeloid Antigen-presenting activated microglia T cell co-stimulation.
Whrn Broad (neuronal bias) Mostly neuronal May be background RNA here.
Pdgfb Endothelial / Pericyte Vascular growth factor Likely paracrine signal to vasculature.
Bcl2l11 (Bim) Broad Apoptosis regulator Expressed in many cell types, including immune.
Ank Broad Cytoskeletal / membrane protein Not specific; low-level across cells.

classic inflammatory myeloid signature, enriched for NF-κB–driven cytokines and co-stimulatory molecules. This cluster is dominated by pro-inflammatory, NF-κB–activated microglia/macrophages, with hallmarks of antigen presentation (Cd86, Tnfsf9), inflammasome activation (Nlrp3, Il1b), and NF-κB–driven cytokines (Tnf, Osm). The Mir155hg / Bcl2a1b/d combination is typical of strong inflammatory activation.

The presence of Pdgfb suggests possible cross-talk to the vascular compartment, which is often seen in perivascular macrophages or activated microglia near blood vessels.

8.2.2.2 Cluster 1

Gene Broad Class Finer Subtype Notes
Tnfaip3 (A20) Myeloid (microglia/macrophage) NF-κB negative feedback in activated microglia Anti-inflammatory regulator.
Ppp1r15a (Gadd34) Myeloid Stress-response myeloid cells Involved in ER stress recovery.
Entpd1 (CD39) Myeloid Microglia with immunoregulatory phenotype Hydrolyzes ATP/ADP to suppress inflammation.
Pde3b Myeloid Lipid metabolism / anti-inflammatory microglia cAMP regulation; linked to alternative activation.
Pag1 Myeloid Lipid raft signaling in immune cells Adapter in immune receptor signaling.
Dapp1 Myeloid PI3K signaling in myeloid cells BCR/TLR pathway adaptor.
Rasgef1b Myeloid Vascular-associated myeloid cells Induced by inflammatory stimuli, linked to endotoxin tolerance.
Agmo Myeloid Lipid metabolism Alkylglycerol monooxygenase; in alternatively activated macrophages.
Casp4 Myeloid Inflammasome-related Non-canonical inflammasome sensor.
Mertk Myeloid Phagocytic / repair microglia Efferocytosis receptor in tissue repair phase.
Dusp6 Myeloid MAPK feedback regulation Dampens ERK signaling in resolution phase.
Tbxas1 Myeloid Eicosanoid metabolism Thromboxane synthase; lipid mediator regulation.
Adrb2 Myeloid Neuroimmune interface microglia β2-adrenergic receptor; responsive to noradrenaline.
Tgm2 Myeloid Wound-healing microglia Cross-links ECM; tissue remodeling.
Fchsd2 Broad Vesicular trafficking Not cell-type specific, enriched in vesicle-rich cells.
Tbc1d9 Broad Endosomal trafficking Not cell-type specific; some enrichment in immune.
Abcd2 Myeloid Lipid transport Peroxisomal transporter in lipid metabolism.
Gpr84 Myeloid Pro-inflammatory switch receptor Medium-chain fatty acid receptor; can be upregulated in inflammation.
Chst8 Broad (secretory cells) Sulfotransferase Low-level in immune.
Myc Broad (proliferating cells) Proliferating microglia/macrophages Transcription factor; cell cycle entry.

anti-inflammatory / resolution-phase myeloid signature. This cluster fits an activated but immunoregulatory / repair-oriented microglia–macrophage phenotype, sometimes called M2-like or resolution-phase microglia.

Key features:

  • Anti-inflammatory feedback: Tnfaip3, Dusp6, Ppp1r15a.
  • Phagocytosis and repair: Mertk, Tgm2.
  • Neuroimmune signaling: Adrb2 (responds to adrenergic input).
  • Metabolic shift: Agmo, Pde3b, Abcd2, Tbxas1.

Compared to cluster 0 (high Tnf, Il1b, Nlrp3), this is a less inflammatory, more resolution-oriented state, possibly spatially closer to degenerating neurons or vasculature undergoing repair.

8.2.2.3 Cluster 2

Gene Broad Class Finer Subtype Notes
Ctsd Myeloid (microglia/macrophage) Lysosomal protease; high in microglia Involved in protein degradation.
Ctsz Myeloid Lysosomal protease; activated microglia Enriched in inflammatory and phagocytic states.
Serpine2 Endothelial / Myeloid ECM remodeling, vascular niches Plasminogen activator inhibitor.
Hif1a Broad (hypoxia-responsive) Hypoxia-adapted microglia/endothelial Master regulator of low-oxygen response.
Fcgr3 (Cd16) Myeloid Inflammatory/activated microglia Fcγ receptor; antibody-dependent activation.
Ctss Myeloid Lysosomal protease; antigen presentation Upregulated in phagocytic microglia.
Grn Myeloid Activated microglia Progranulin; injury/inflammation marker.
Hexb Myeloid Homeostatic microglia marker Core microglia identity gene.
Baiap2l2 Broad Cytoskeletal protein Low cell-type specificity; may relate to motility.
Egln3 Broad (hypoxia response) Oxygen sensor enzyme Part of HIF pathway regulation.
P4ha1 Broad (fibrosis / ECM) ECM collagen hydroxylation Often induced in hypoxic environments.
Pfkfb3 Broad (glycolytic switch) Hypoxia-activated microglia Key glycolysis regulator; metabolic adaptation.
Rab7b Myeloid Endo-lysosomal trafficking High in phagocytic microglia/macrophages.
Gpr183 (Ebi2) Myeloid Chemotactic receptor Guides migration towards oxysterol gradients; seen in microglia.
Adarb1 Broad (neurons & glia) RNA editing enzyme Some microglial expression.
Gcnt2 Broad (glycosylation) Glycan biosynthesis Low specificity; in immune/vascular.
Gm56614 Unknown Predicted gene Likely co-expressed with myeloid genes.
H1f3 Broad (proliferating cells) Histone variant H1.3 Chromatin regulation; not specific.
Tagap Myeloid / Lymphoid T cell and microglial activation gene Rho GTPase-activating protein.
Gm57375 Unknown Predicted gene Likely co-expressed with immune/hypoxia genes.

Hypoxia- and lysosome-enriched myeloid state, possibly linked to perivascular or hypoxic microglia/macrophages. Cluster 2 has a strong lysosomal–phagocytic core (Ctsd, Ctss, Ctsz, Rab7b, Hexb), combined with hypoxia/metabolic adaptation genes (Hif1a, Egln3, Pfkfb3, P4ha1). This is typical of microglia or perivascular macrophages in hypoxic environments (e.g., around ischemic lesions or tightly packed tumor vasculature).

The presence of Serpine2 and P4ha1 suggests ECM remodeling and perivascular interaction, while Fcgr3 and Tagap keep the profile immune-competent.

8.2.2.4 Cluster 4

8.2.2.5 Cluster 1

homeostatic microglia–enriched cluster, with some immune trafficking receptors and neuronal-interaction genes.

Gene Broad Class Finer Subtype Notes
Gm10790 Unknown Predicted transcript Likely co-expressed with microglial genes.
8030442B05Rik Unknown Predicted transcript Expression pattern unclear; may follow microglial clusters.
P2ry12 Microglia Homeostatic microglia Canonical marker; downregulated in activation.
Gm2629 Unknown Predicted transcript Likely microglial co-expression.
Ccr5 Myeloid (microglia/macrophage) Chemotactic receptor Inflammatory chemokine receptor; can be expressed in microglia and infiltrating monocytes.
Siglech Microglia / pDC (rare in brain) Border-associated microglia signature In brain, more linked to microglial subtypes.
A830008E24Rik Unknown Predicted transcript Likely microglia-associated.
Zfhx3 Broad (neurons & glia) Transcription factor Regulates circadian and neuronal genes; low-level in microglia.
4933406I18Rik Unknown Predicted transcript Likely co-expressed in microglial niche.
Srgap2 Neuron / Microglia interaction Axon guidance-related; microglia express in synaptic pruning Implicated in neuron–microglia synapse remodeling.
Atp8a2 Neuron Phospholipid flippase Mostly neuronal; may appear from neuronal RNA uptake.
Ophn1 Neuron Synaptic Rho-GTPase regulation Mostly neuronal expression.
Csmd3 Neuron Synaptic adhesion molecule Likely neuronal background RNA in cluster.
Tgfbr1 Broad (microglia, astrocytes, endothelium) TGF-β receptor Important for homeostatic microglia maintenance.
Dock8 Myeloid Cytoskeletal remodeling in immune cells Linked to motility and immune synapses.
Ltc4s Myeloid (microglia/macrophage) Leukotriene synthesis Eicosanoid metabolism; inflammatory signaling.
Crybb1 Broad (lens/rare brain) Crystallin βB1 Likely low-level or background.
P3h2 Broad (collagen metabolism) Prolyl 3-hydroxylase Not cell-type specific.
Fhit Broad Tumor suppressor Low specificity.
E330020D12Rik Unknown Predicted transcript Likely low-abundance.

The anchor gene here is P2ry12, marking this as a homeostatic microglia cluster, likely representing surveillant microglia in healthy or less perturbed brain regions.

Supporting markers for microglial identity:

  • Tgfbr1 → required for microglial homeostasis.
  • Siglech, Dock8, Ltc4s → immune cell motility/communication genes.

The presence of multiple neuronal genes (Atp8a2, Ophn1, Csmd3, Srgap2) is probably due to phagocytosis of neuronal debris or ambient RNA contamination, which is common in microglial clusters.

8.2.2.6 Cluster 6

This cluster presents a strong disease-associated microglia / phagocytic macrophage profile, often seen in neurodegeneration, demyelination, or chronic injury models.

Gene Broad Class Finer Subtype Notes
Gpnmb Microglia / Macrophage DAM (disease-associated microglia) Lysosomal membrane protein; neurodegeneration marker.
Igf1 Microglia Neuroprotective / repair microglia Growth factor promoting neuronal survival.
Lgals3 (Galectin-3) Microglia / Macrophage DAM / activated phagocytic microglia Involved in phagocytosis and inflammation.
Lilrb4a Myeloid Immune checkpoint receptor Regulatory macrophage/microglia.
Spp1 (Osteopontin) Microglia / Macrophage DAM / chronic inflammation Cytokine–ECM glycoprotein; high in aged or diseased microglia.
Cd63 Broad (immune/vesicular) Exosome/lysosome protein Enriched in phagocytic microglia.
Ftl1 Broad (iron metabolism) Ferritin light chain Iron storage; upregulated in activated microglia.
Cd68 Microglia / Macrophage Lysosomal marker Pan-phagocytic myeloid cell marker.
Ms4a7 Myeloid Macrophage marker More common in perivascular macrophages, but present in DAM.
Atp6v0d2 Myeloid Lysosomal acidification Part of vacuolar ATPase; linked to osteoclast-like microglia.
Hectd2os Unknown Non-coding transcript Co-expressed with inflammatory myeloid genes.
Itga5 Myeloid ECM adhesion receptor Fibronectin receptor; motile microglia.
Fabp5 Myeloid Lipid metabolism Fatty acid–binding protein; enriched in activated microglia.
Adarb1 Broad RNA editing enzyme Low-level in microglia.
Lyz2 Myeloid Pan-myeloid lysosomal enzyme High in macrophages, microglia, neutrophils.
Fth1 Broad Iron metabolism Ferritin heavy chain; iron storage.
Atosa Unknown Predicted transcript Likely myeloid-associated.
Rgs16 Myeloid GPCR signaling regulator Involved in chemotaxis control.
Ell2 Broad (immune) Transcription elongation Present in plasma cells and some myeloid cells.
Mmp12 Myeloid ECM-degrading macrophage/microglia Elastase; high in infiltrating macrophages and activated microglia.

Cluster 6 is a phagocytic, lipid-metabolizing, lysosome-rich microglial/macrophage population, with hallmark DAM genes (Gpnmb, Lgals3, Spp1, Cd68, Cd63, Fabp5) and repair-associated factors (Igf1, Lilrb4a). The iron storage genes (Ftl1, Fth1) point to involvement in iron homeostasis following injury or myelin debris clearance.

8.2.2.7 Cluster 8

This cluster´s profile fits an early-activation / stress-response microglia state, with transcription factors and immediate early genes suggesting a recently stimulated but not yet fully inflammatory phenotype.

Gene Broad Class Finer Subtype Notes
Atf3 Myeloid (microglia/macrophage) Stress-response / injury-induced microglia Immediate early transcription factor.
Dusp1 Myeloid MAPK signaling feedback Negative regulator of ERK/JNK signaling; early activation marker.
Cd83 Myeloid Antigen-presenting microglia Immune co-stimulatory protein; often induced by TLR stimulation.
Ier5 Broad (stress-response) Immediate early response gene Transcriptional regulator in acute stress.
Csf1 Myeloid Growth factor for microglia/macrophages Promotes survival and proliferation; autocrine in microglia.
Ptchd1 Broad (neuronal bias) Developmental regulator Likely low-level neuronal RNA.
Runx1 Myeloid Development/activation TF Key in microglial development and activation.
Mylip Broad Lipid metabolism / ubiquitination Ubiquitin ligase with metabolic regulation role.
Rgs2 Myeloid GPCR signaling regulator Often transiently induced during microglial activation.
Basp1 Broad Cytoskeletal / signaling Weakly enriched in immune and neuronal cells.
Mafb Myeloid Transcription factor for differentiated microglia Maintains mature identity; can be induced during repair.
Mir142hg Myeloid Host gene for miR-142 Enriched in immune cells; regulates activation state.
Arhgap22 Myeloid Cytoskeletal regulation Rho GTPase–activating protein in immune cells.
Gm26542 Unknown Predicted transcript Likely co-expressed with immune genes.
Gm35417 Unknown Predicted transcript Likely immune-linked.
Creb5 Myeloid cAMP response TF Involved in immune modulation and differentiation.
Ifitm10 Broad (immune bias) IFN-inducible transmembrane protein Lowly expressed ISG.
Ly86 (MD-1) Myeloid TLR4/MD-2 co-receptor partner Enriched in microglia and B cells; innate sensing.
H1f2 Broad Histone H1.2 Cell cycle/chromatin; not specific.
Sgk1 Myeloid Stress-activated kinase Regulates survival and inflammatory gene expression.

Cluster 8 appears to be a transient, early-activated microglial state, positioned between homeostatic and fully inflammatory profiles. Hallmarks include:

  • Immediate early / stress genes: Atf3, Dusp1, Ier5, Rgs2.
  • Activation TFs: Runx1, Mafb, Creb5.
  • Immune co-stimulatory signals: Cd83, Ly86.
  • Growth factor support: Csf1.

This cluster might represent microglia that have recently sensed a stimulus (e.g., DAMPs, cytokines) and are in the priming phase before committing to an inflammatory, DAM, or resolution phenotype.

8.2.2.8 Cluster 10

This cluster has a antigen-presenting, Spp1⁺ disease-associated microglia/macrophage state with strong MHC-II expression and co-stimulatory capacity.

Gene Broad Class Finer Subtype Notes
H2-Aa Myeloid (microglia/macrophage) MHC-II antigen-presenting microglia Core MHC-II α-chain.
H2-Eb1 Myeloid MHC-II antigen-presenting microglia MHC-II β-chain.
H2-Ab1 Myeloid MHC-II antigen-presenting microglia MHC-II β-chain.
Cd74 Myeloid MHC-II chaperone / invariant chain Required for MHC-II trafficking.
Itgax (CD11c) Myeloid Activated antigen-presenting microglia Common in DAM and inflammatory microglia.
Spp1 (Osteopontin) Myeloid DAM / chronic activation Cytokine–ECM protein, high in aged/diseased microglia.
Ccl6 Myeloid Inflammatory chemokine Attracts monocytes/macrophages.
Cst7 Myeloid Cytotoxic / phagocytic microglia Cystatin F, lysosomal protease inhibitor.
Atp6v0d2 Myeloid Lysosomal acidification Also in cluster 6; osteoclast-like microglia.
Dkk2 Myeloid DAM / Wnt pathway modulator Seen in neurodegenerative microglia.
Ctsl Myeloid Lysosomal protease Cathepsin L; phagocytic microglia.
B2m Broad (immune) MHC-I and MHC-II component β2-microglobulin.
Ctnna3 Broad (neuronal adhesion) Likely neuronal RNA background Uncommon in microglia.
Fam20c Broad (secretory) Protein kinase for ECM proteins Limited immune specificity.
Otud1 Myeloid Ubiquitin processing Regulates inflammatory signaling.
Fcrls Microglia Homeostatic microglia marker Microglia-specific Fc receptor–like scavenger.
Cxcl9 Myeloid IFN-γ–induced chemokine Recruits Th1 T cells.
Tlr2 Myeloid Innate immune receptor Recognizes bacterial/fungal products.
Tgfb1 Myeloid Regulatory cytokine Maintains microglia homeostasis; immune suppression.
Postn (Periostin) Broad (ECM) ECM remodeling Often upregulated near vasculature or in reactive stroma.

Cluster 10 represents antigen-presenting microglia/macrophages with strong MHC-II expression (H2-Aa, H2-Ab1, H2-Eb1, Cd74), co-stimulatory marker Itgax, and inflammatory chemokines (Ccl6, Cxcl9). The combination of Spp1, Dkk2, Atp6v0d2, Cst7 points toward Spp1⁺ DAM, often found in chronic neuroinflammation, neurodegeneration, or demyelination.

Compared to the other DAM-like cluster (6), this one has:

  • Stronger antigen presentation signature (MHC-II, Cd74, Itgax)
  • More IFN-γ–responsive chemokines (Cxcl9)
  • Retains some homeostatic microglia marker (Fcrls)

8.2.2.9 Cluster 12

This cluster looks like a Gpr34⁺ / C3ar1⁺ homeostatic-to-early-activated microglia profile, with some neuronal-adhesion transcripts likely from ambient RNA.

Gene Broad Class Finer Subtype Notes
Fgf13 Broad (neuronal bias) Neuronal cytoskeletal regulator Likely background from neurons.
Gm15155 Unknown Predicted transcript Co-expressed with immune genes.
Plxdc2 Broad (endothelial / neuronal) ECM receptor Expressed at neurovascular interface.
Cacna1a Neuron P/Q-type calcium channel Strong neuronal background RNA.
Or5v1b Olfactory receptor family Likely not functional in brain Probably low-level mis-mapping.
Nrp1 Broad (neurons, endothelial, immune) Axon guidance / immune migration In microglia, linked to angiogenesis and repair.
Apbb1ip Myeloid Cytoskeletal and adhesion regulator Promotes immune cell migration.
Arhgap24 Broad (neurons & immune) Rho GTPase–activating protein Cytoskeleton and migration.
Lrmda Broad (neuronal development) Retinal/neuronal differentiation factor Likely background.
Fyb (ADAP) Myeloid / Lymphoid Immune synapse adaptor T cell and microglia activation.
Tanc2 Neuron Synaptic scaffold protein Likely neuronal RNA background.
Gpr34 Microglia Homeostatic microglia marker Strong identity gene for surveillant microglia.
C3ar1 Microglia / Macrophage Complement receptor Microglial chemotaxis and phagocytosis.
Nav2 Broad (neurons) Neuronal migration Background RNA.
Sdk1 Neuron Synaptic adhesion molecule Likely ambient neuronal RNA.
Gm56614 Unknown Predicted transcript Also appears in your hypoxia cluster (2).
Wwox Broad (tumor suppressor) Ubiquitous No cell-type specificity.
Marchf3 Myeloid E3 ubiquitin ligase Found in immune regulation.
Immp2l Broad (mitochondrial) Mitochondrial peptidase Housekeeping.
Plcl1 Neuron Phospholipase C-like protein Strong neuronal bias.

Clusters key microglia markers are Gpr34 and C3ar1, both associated with homeostatic or early-activated microglia. Several genes (Nrp1, Apbb1ip, Arhgap24, Fyb, Marchf3) point to motility and immune synapse formation, suggesting a migratory/interactive state.

However, there’s substantial neuronal RNA background (Cacna1a, Tanc2, Sdk1, Nav2, Fgf13), which could be due to microglia physically interacting with neurons or to ambient RNA contamination in single-cell prep.

Compared to the “resting-like” cluster (4, P2ry12⁺), this one:

  • Shares homeostatic markers but
  • Shows more motility-related genes and complement receptor C3ar1 (engaged in surveillance or early response).

8.2.2.10 Cluster 27

This cluster seems a proliferating / cell-cycle microglia cluster, with strong mitotic spindle and chromosome segregation gene expression.

Gene Broad Class Finer Subtype Notes
Birc5 (Survivin) Proliferating cells (microglia) G2/M-phase marker Anti-apoptotic and mitotic regulator.
Cdca3 Proliferating cells Cell division cycle protein Regulates G1/S transition.
Cdca8 Proliferating cells Chromosomal passenger complex Essential for mitosis.
Mki67 Proliferating cells Pan-cell-cycle marker Gold standard proliferation marker.
Tpx2 Proliferating cells Mitotic spindle assembly Microtubule-associated.
Cenpe Proliferating cells Kinetochore motor protein Chromosome alignment during mitosis.
Cenpf Proliferating cells Kinetochore function Required for proper mitosis.
Knl1 Proliferating cells Kinetochore scaffold protein Essential for chromosome segregation.
Cenpa Proliferating cells Centromere-specific histone H3 variant Marks active centromeres.
Ube2c Proliferating cells Ubiquitin-conjugating enzyme Cell cycle progression.
Top2a Proliferating cells DNA topoisomerase Required for DNA replication/mitosis.
Hmmr Proliferating cells Spindle positioning Hyaluronan-mediated motility receptor.
Nusap1 Proliferating cells Spindle assembly protein Stabilizes microtubules in mitosis.
H3c3 Proliferating cells Histone H3.3 Chromatin assembly in dividing cells.
Prc1 Proliferating cells Cytokinesis protein Central spindle formation.
Aspm Proliferating cells Mitotic spindle pole protein Linked to neurogenesis and cell division.
Cdkn1a (p21) Proliferating cells Cell cycle checkpoint Can indicate DNA damage or repair pause.
Rrm2 Proliferating cells Ribonucleotide reductase dNTP synthesis for DNA replication.
Ifi27l2a Myeloid / interferon response ISG; possible immune activation overlay Not core proliferation marker.
Gm42047 Unknown Predicted transcript Likely co-expressed in dividing cells.

Cell-cycle cluster, almost certainly representing dividing microglia/macrophages in the dataset. These cells express:

  • Core mitotic machinery (Mki67, Top2a, Birc5, Cenpe/f, Tpx2, Nusap1).
  • DNA replication enzymes (Rrm2, H3c3).
  • Cytokinesis regulators (Prc1, Aspm).

These are likely microglia proliferating in response to injury or inflammation, possibly bridging from homeostatic/early activation states into DAM or antigen-presenting states.